Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Seminiferous tubule degeneration and infertility in mice with sustained activation of WNT/CTNNB1 signaling in sertoli cells

ABSTRACT: To elucidate the role(s) of WNT/CTNNB1 signaling in the testis, transgenic Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ mice were generated to obtain sustained activation of the WNT/CTNNB1 pathway in Sertoli cells. Male Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ mice were sterile because of testicular atrophy starting at 5 wk of age, associated with degeneration of seminiferous tubules and the progressive loss of germ cells. Sustained WNT/CTNNB1 pathway activation was obtained in Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ Sertoli cells. Sertoli cells often exhibited morphological characteristics suggestive of incomplete differentiation that appeared in a manner coincident with germ cell loss, accompanied by an increase in the expression of the immature Sertoli cell marker AMH. Microarray analyses performed on cultured Sertoli cells showed that CTNNB1 induces the expression of genes associated with the female sex determination pathway as well as cell-cycle associated genes . Together, these data suggest that the WNT/CTNNB1 pathway regulates Sertoli cell functions critical to their capacity to support spermatogenesis and to proliferate in the postnatal testis. Sertoli cells were obtained from 3wks-old Ctnnb1(tm1Mmt/tm1Mmt) mice. Cells were infected with adenoviruses to express eGFP or Cre in serum-free medium, and subsequently harvested for RNA extraction. Preliminary experiments demonstrated that an infection efficiency of nearly 100% could be obtained at an MOI of 50 (as determined by analysis of fluorescent signal in Ad-eGFP-infected cells) and that the Ad-Cre virus produced complete recombination of the floxed Ctnnb1 allele after 12 hours. Microaaray analyses were done using triplicate RNA samples from each adenoviral treatment using MouseRef-6 v.2.0 expression BeadChips technology (Illumina, San Diego, CA).

ORGANISM(S): Mus musculus

SUBMITTER: Derek Boerboom

PROVIDER: E-GEOD-28402 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To study the physiological role of WNT4 in the postnatal ovary, a mouse strain bearing a floxed Wnt4 allele was created and mated to the Amhr2tm3(cre)Bhr strain to target deletion of Wnt4 to granulosa cells. Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice had significantly reduced ovary weights and produced smaller litters (P<0.05). Serial follicle counting demonstrated that, while Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice were born with a normal ovarian reserve and maintained normal numbers of small follicles until puberty, they had only 25.2% of the normal number of healthy antral follicles. Some Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice had no antral follicles or corpora lutea and underwent premature follicle depletion. RTPCR analyses of Wnt4flox/-;Amhr2tm3(cre)Bhr/+ granulosa cells and cultured granulosa cells that overexpress WNT4 demonstrated that WNT4 regulates the expression of Star, Cyp11a1 and Cyp19, steroidogenic genes previously identified as downstream targets of the WNT signaling effector CTNNB1. WNT4- and CTNNB1-overexpressing cultured granulosa cells were analyzed by microarray for alterations in gene expression, which showed that WNT4 also regulates a series of genes involved in late follicle development and the cellular stress response via the WNT/CTNNB1 signaling pathway. Together, these data indicate that WNT4 is required for normal antral follicle development, and may act by regulating granulosa cell functions including steroidogenesis. Experiment Overall Design: Granulosa cells were obtained from 20-26 day-old mice of various genotypes 48h after eCG treatment, using the needle puncture method as previously described. Cells were then infected with adenoviruses to express eGFP or Cre, or overexpress WNT4 in serum-free medium, and subsequently harvested for RNA extraction as described below. Preliminary experiments demonstrated that an infection efficiency of >80% could be obtained at an MOI of ~50 (as determined by analysis of fluorescent signal in Ad-eGFP-infected cells) and that the Ad-Cre and Ad-WNT4 viruses produced efficient recombination of the floxed Ctnnb1 alleles and robust WNT4 overexpression, respectively. Microarray analyses were done using triplicate RNA samples from each adenoviral treatment described above, and using mouse expression set 430 microarrays (Affymetrix, Santa Clara, CA).

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Seminiferous tubule degeneration and infertility in mice with sustained activation of WNT/CTNNB1 signaling in sertoli cells

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